A novel, double-stranded DNA-activated protein kinase (DNA-PK) has been extensively purified from HeLa cells. Several monoclonal antibodies have been made which specifically inhibit the enzyme's activity and recognize a single 350 kDa polypeptide on Western blots. DNA-PK will be purified to homogeneity using FPLC and immunoaffinity chromatography, and its unique biochemical properties will be studied. Interaction of the enzyme with DNA will be probed by characterizing its requirements for primary sequence and secondary structure of the activating polynucleotide. Specific reagents and assays will be developed, including additional monoclonal and polyclonal antibodies, synthetic peptide substrates, and nucleic acid probes. The latter will be molecularly cloned using cDNA expression libraries probed with monoclonal antibodies. These reagents will be used to determine the interspecies distribution and intracellular localization of DNA-PK, and also to study the regulation of DNA-PK expression at the genetic level. The biologic function(s) of DNA-PK will be elucidated by examining (l) its interactions with other cellular and viral proteins, using co-immunoprecipitation and Western blotting; and (2) its presence and activity at different stages of the cell cycle using synchronous populations of HeLa cells obtained by centrifugal elutriation and by metabolic inhibition. Because of the extraordinary activating effect of double-stranded DNA on the phosphorylation of substrate proteins by DNA-PK, the site of action of DNA-PK within the cell is likely to be chromatin or proteins that interact with chromatin. Understanding the molecular structure, mechanism and function of this heretofore unknown type of protein kinase could therefore have major significance for understanding the regulation of gene activity and/or chromatin dynamics, including the molecular processes associated with DNA replication, recombination, DNA repair, and cell division. Thus the biological function of DNA-PK and/or related protein kinases could impact a variety of human diseases, including neoplasia, developmental abnormalities, and the response to viral infection.